biologic drug delivery across the blood-brain barrier with ......biologic drug delivery across the...
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Biologic Drug Delivery
Across the
Blood-Brain Barrier
with IgG Fusion Proteins
William M. Pardridge, M.D.
University of California
ArmaGen, Inc.
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The Brain and Biotechnology
In 2017, there are no
biologics that are FDA
approved for CNS disease,
where the drug crosses the
blood-brain barrier
(BBB)
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Brain Uptake of Therapeutic Antibodies:
The myth of a brain uptake of 0.1-0.2%
A frequently cited claim is that the brain uptake of
therapeutic antibodies is low but significant, and the
brain antibody concentration is
0.1-0.2% of the serum antibody concentration.
The evidence for this claim is the observation that the
CSF concentration is 0.1-0.2% of the serum
concentration of the antibody.
This assumes that drug entry into CSF, across the
blood-CSF barrier at the choroid plexus, is equal to
drug entry into brain parenchyma, across the blood-
brain barrier, at the brain capillary endothelium.
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capillary endothelium
IgG CSF to
serum ratio
=
0.1-0.2%
Blood to CSF vs
Blood to Brain
IgG brain to
serum ratio
=
<0.01%
Therapeutic
Antibody
in Blood
Blood-
CSF
Barrier
@
Choroid
Plexus
CSF
Blood-
Brain
Barrier
@
Capillary
Endothe-
lium
Brain
The
blood-CSF
barrier
is 100-fold
leakier than
the BBB.
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brain
spinal cord
The Blood-Brain Barrier
~2% of all small
molecule drugs
cross the BBB
~0% of all large
molecule drugs
cross the BBB
6median eminence (CVO)
Intravenous HRP is trapped in
the lumen of brain capillaries
(Brightman)
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blood
brain
red cell
GLUT1
luminal
membrane
GLUT1
abluminal
membrane
Intra-endothelial
volume
0.8 uL/gram
Extra-vascular
volume
700 uL/gram
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Endogenous BBB Transporters
Nutrient Transport:
Carrier-Mediated Transport
Peptide Transport:
Receptor-Mediated Transport
GLUT1MCT1
LAT1
CAT1
CNT2
NBT ?
? CTL1
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Immune-staining of capillaries in brain with
an antibody to a BBB peptide receptor
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Human and primate BBB insulin receptor:Mediates transport of insulin and an insulin receptor antibody
Human brain capillaries
Emulsion auto-radiography of
rabbit brain after carotid
artery infusion of [125I]-insulin
Immunocytochemistry of Rhesus monkey brain
with MAb against human insulin receptor (HIR)
Brain scanning in Rhesus monkeys
Peptide alone Peptide-HIRMAb
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Chimeric or
humanized
HIRMAb
+
Recombinant
protein
=
IgG fusion protein:a New Biological Entity
IgG Fusion Proteins for Delivery of Protein
Therapeutics to the Human Brain
13 BBB
Neuron
lysosome
Blood Brain interstitium
R NTR
MAb-neurotrophin
TNFaMAb-decoy receptor
R
MAb-enzyme
RR
amyloidMAb-therapeutic antibody
R
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AGT-181: HIRMAb-IDUA fusion proteinA BBB-penetrating form of iduronidase (IDUA)
HIRMAb-IDUA
fusion protein
HIRMAb
domain
IDUA
domain
HIRMAb=monoclonal antibody (MAb) to human insulin receptor (HIR)
AGT-181
Intracellular
delivery of
AGT-181 to
lysosomes of Hurler
fibroblastsIDUA
alone
AGT-
181
Brain uptake in the
Rhesus monkey
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70% reduction in brain HS
Reduction of glycosaminoglycans (GAGs) in brain of
MPS mice with systemic administration of IgG-
lysosomal enzyme fusion proteins
Hurler mouse (MPSI)
• 6 month old MPSI mice
• null for iduronidase (IDUA)
• treat twice weekly (1 mg/kg)
IV for 6 weeks
• measure brain GAGs by light
and electron microscopy
cTfRMAb-IDUAsaline
Treatment causes a 73%
reduction in brain
lysosomal inclusion bodies
• 2 week old MPSIIIA mice
• null for sulfamidase (SGSH)
• treat thrice weekly (5 mg/kg)
IP for 6 weeks
• measure brain and liver
Heparan Sulfate (HS) by LC-MS
Sanfilippo A mouse (MPSIIIA)
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First BBB Trojan Horse Clinical Trials
(INDs approved 2015)
Mucopolysaccharidosis Type I
Hurler Syndrome
HIRMAb-IDUA fusion protein
Valanafusp alpha
(AGT-181)
IDUA = iduronidase
Mucopolysaccharidosis Type II
Hunter Syndrome
HIRMAb-IDS fusion protein
(AGT-182)
IDS = iduronate 2-sulfatase
The Mucopolysaccharidoses (MPS),
Lysosomal storage disorders
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BBB-
penetrating
decoy
receptors
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CH2
CH3
TNFR-II
TNFa VH
VL
CL
CH1
CH2
CH3
TNFR-II
TNFa
BBB
etanercept
Re-engineering the TNFR2 extracellular domain
as a BBB-penetrating IgG fusion protein
BBB-penetrating
etanercept:
HIRMAb-TNFR2
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Brain uptake in the Rhesus monkey:Etanercept (Fc:TNFR) vs HIRMAb-TNFR fusion protein
Rhesus monkey
(1) Brain uptake of entanercept (Fc:TNFR) is confined to brain plasma volume
(1)
(2)
(2) Brain uptake of HIRMAb-TNFR, 3% ID/brain, compares to small molecules
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Therapeutic effects in mouse models of neural disease:
Etanercept vs BBB-penetrating-TNFR2 fusion protein
Parkinson’s
disease
Striatal tyrosine
hydroxylase (TH) after
6-hydroxydopamine
toxin exposure
Alzheimer’s disease
Immunoreactive Abeta amyloid
plaque in PSAPP double
transgenic AD mouse
stroke
Dye staining of mouse
brain shows infarct after
60 min reversible
occlusion MCA
IV TfRMAb-TNFR2
reverses motor deficit
by 80% and increases
striatal TH enzyme
activity by 130%
IV Enbrel has no effect
IV TfRMAb-TNFR2
reduces stroke
volume by 50% and
improves neural
deficit by 50%
IV Enbrel has no
effect
IP TfRMAb-TNFR2
reduces total plaque
and brain Ab1-42,
reduces ICAM-1 and
improves recognition
memory
IP Enbrel has no effect
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BBB-Penetrating
Bi-Specific Antibodies
Targeting Abeta
Amyloid in Alzheimer’s
Disease
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Re-engineering an anti-Ab antibody as a
BBB-penetrating tetravalent bi-specific antibody (BSA)
Brain uptake =
<0.01% ID/brain
Anti-Ab MAb
amyloid
Brain uptake =
2% ID/brain
Anti-BBB MAb
BBB receptor
Anti-BBB/anti-Ab BSA
Brain uptake =
2% ID/brain
Anti-Ab ScFv
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Chronic treatment of AD transgenic mouse with
BBB-penetrating bi-specific antibody (BSA)
APPswe, PSEN1dE9
double transgenic
Alzheimer’s disease (AD)
mouse
Methods
1. Treat AD mice with either saline or 5 mg/kg BBB-penetrating
anti-Ab BSA administered subcutaneously (SQ)
2. Treat daily for 12 weeks (mice 12-15 months old)
3. Measure brain amyloid plaque with 6E10 immune labeling and
Thioflavin-S in cortex and hippocampus
4. Quantify immune response with anti-drug antibody (ADA)
ELISA
5. Measure cerebral micro-hemorrhage with Prussian blue
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0
1
2
3
4
5
1 2
saline
Anti-Ab
BSA
Brain amyloid plaque (%)
Amyloid plaque reduction in AD mouse brain with a
BBB-penetrating anti-Ab BSA
60% reduction in brain
amyloid plaque with daily
SQ administration of BBB-
penetrating anti-Ab BSA
(5 mg/kg) for 12 weeks
Anti-Ab
BSA
Cortex
6E10
Saline
Cortex
6E10
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Neuro-
trophins:
EPO
GDNF
Lysosomal
enzymes:
IDUA
IDS
ASA
SGSH
NAGLU
Decoy
Receptors:
TNFR-II
Therapeutic
antibodies:
Anti-Ab MAb
Human BBB Trojan horse fusion proteins:Re-engineering protein therapeutics for delivery to human brain
27Clinical Pharmacology & Therapeutics (April, 2015)